DEAD-box proteins are superfamily 2 RNA helicases that are required for virtually every process carried out by structured RNAs, from pre-mRNA splicing and translation to intracellular trafficking of proteins and RNAs. They are also required for replication of viruses including HIV-1 and HCV and overexpression is linked to colon and prostate cancer. These proteins use energy from ATP binding and hydrolysis to facilitate RNA conformational changes and folding transitions, but we have limited molecular knowledge about how they manipulate RNA structure. Insights from mechanistic studies will be critical for deep understanding of fundamental biological processes and for understanding and ultimately treatment of important viral diseases and cancer. We have focused on the fungal DEAD-box proteins CYT-19 and Mss116p, which function as general RNA chaperones in folding of mitochondrial group I and group II self-splicing introns. These systems are powerful for mechanistic studies because the RNAs are relatively simple and tractable, and their catalytic activity provides a robust and sensitive readout for formation of the native state. In years 1-5 of funding (2004-2009), we used a well-studied group I intron from Tetrahymena thermophila to show that CYT-19 functions as a true chaperone, facilitating refolding of a long-lived misfolded conformation without functioning in the downstream catalytic steps. We found that CYT-19 disrupts structure non-specifically, without distinguishing native from misfolded conformations, and this activity favors accumulation of the native RNA because it is much more stable than the misfolded conformation. Since submitting a renewal that received two years of ARRA funding (2009-2011), we made further advances. Single molecule fluorescence and rapid kinetics showed directly that CYT-19 can be strongly inhibited by tertiary structure, unwinding a short helix only after the helix spontaneously 'undocks' from tertiary contacts with the intron core. Rapid kinetics experiments also supported a model in which a C-terminal 'tail' of CYT-19 contacts structured RNA and tethers the helicase core to disrupt nearby structure, and a series of rate measurements indicated that CYT-19 completely unwinds short RNA helices in a single cycle of ATP- dependent conformational changes. Together, the work leads to a general model for RNA chaperone activity by DEAD-box proteins in which the proteins are localized to structured RNAs by tethering, and they disrupt exposed elements of secondary structure non-processively to allow these segments an opportunity to form new contacts. Here we propose to test and extend this model in two important directions. In Aims 1 and 2, we will use simple duplex substrates and an arsenal of experimental approaches to probe how the ATPase cycle is coupled to RNA unwinding and to what extent tethering constrains the position and orientation of the helicase core. In Aims 3 and 4 we will use physical and chemical approaches to follow folding of group I and group II introns that rely on CYT-19 and Mss116p in vivo, testing specific hypotheses and probing whether our model for general chaperone activity describes the effects of these proteins on folding of their cognate RNAs.